1. Field of the Invention
The present invention relates to a scanning optical microscope.
2. Description of the Related Art
A scanning laser microscope in which, an acoustic optical element is used has hitherto been known. The acoustic optical element is an element in which, an acoustic optical effect is used. When an acoustic wave is propagate through a solid or a liquid, a periodic fluctuation in a refractive index occurs in that medium due to a photoelastic effect. This periodic fluctuation occurs in parallel to a direction of propagation of the acoustic wave. Moreover, in this periodic fluctuation, a cycle of fluctuation is a wavelength of the acoustic wave. As light is incident on the medium, apart of the light incident is diffracted by an ultrasonic sound wave. This phenomenon is called as the acoustic optical effect.
A principle of operation of the acoustic optical element (such as AOM (acoustic optical modulator), AOTF (acoustic optical tunable filter, and AOD (acoustic optical deflector)) is shown in FIG. 7. In the acoustic optical element, optical crystal such as LiNbO3, PbMoO4, or TeO2 is used as a medium. A transducer that transmits ultrasonic sound waves is attached to the optical crystal. A piezoelectric body is used as the transducer. As a high-frequency voltage (RF (radio frequency) voltage) is applied to the transducer, acoustic waves of high frequency are generated in the crystal. Incident light is caused to be diffracted by using a periodic change of the refractive index due to the acoustic waves, and accordingly, light is caused to be deflected.
The entire incident light that is incident on the acoustic optical element is not diffracted, and first-order diffracted light and transmitted light (non-diffracted light) emerge from the acoustic optical element. By changing the frequency of the high-frequency voltage applied to the acoustic optical element, a direction of deflection of the first-order diffracted light is changed, and by changing amplitude of the high-frequency voltage, a light intensity of the first-order diffracted light changes.
As a scanning laser microscope in which such acoustic optical element is used, a scanning laser microscope disclosed in Japanese Patent Publication No. 4729269 is available.
In the scanning laser microscope disclosed in Japanese Patent Publication No. 4729269, the acoustic optical element is used for modulation of the light intensity. In this scanning laser microscope, the acoustic optical element is disposed between a laser light source and a laser scanning section.
As one of applications in the scanning laser microscope, light stimulus is applied to an observed object (generally, a living cell or a tissue), and a reaction with respect to the light stimulus is subjected to imaging, or an electric potential of the cell is measured. In light stimulus, light of various intensities is irradiated to the observed object.
Moreover, as another application, a portion from a surface to an interior of the observed object is subjected to imaging continuously, and a three-dimensional image is generated from continuous images acquired. In a case of acquiring continuous images, deeper the position in the interior portion of the observed object, at which an image is to be acquired, more is the attenuation of laser light. In order to be able to acquire images of uniform brightness at any position at which the image is acquired, it is necessary to change the light intensity of laser light according to the position at which the image is to be acquired. In view of such circumstances, in the application in which, the light stimulus is to be applied or in the application in which, the three-dimensional image is to be generated, the light intensity is changed, or in other words, modulation of the light intensity is carried out.
In a scanning laser microscope to be used in such applications, an acoustic optical element is used for carrying out modulation of the light intensity as in Japanese Patent Publication No. 4729269. An example of a conventional scanning laser microscope is shown in FIG. 8A and FIG. 8B. FIG. 8A is a diagram showing an arrangement of the scanning laser microscope, and FIG. 8B is a diagram showing laser light at a position of a pupil of an objective.
As shown in FIG. 8A, the scanning laser microscope includes a laser light source 101, an acoustic optical element 102, an optical scanner 103, a pupil projection lens 104, an image forming lens 105, a dichroic mirror 106, a microscope objective 107, and a photodetector 109. If necessary, an excitation filter may be disposed in an optical path from the laser light source 101 up to the optical scanner 103, and a barrier filter (absorption filter) may be disposed in an optical path from the dichroic mirror 106 up to the photodetector 109. Broken-line arrow marks will be described later.
The laser light source 101 is an ultrashort pulse laser for instance. Near-infrared laser light is emitted from the ultrashort pulse laser. Laser light emitted from the laser light source 101 is incident on the acoustic optical element 102. Laser light (first-order diffracted light) emerged from the acoustic optical element 102 is incident on the optical scanner 103. The optical scanner 103 includes two light deflecting elements. The light deflecting element is a galvano scanner for instance. The laser light is deflected in X-direction at a mirror of one of the galvano scanners, and is deflected in Y-direction at a mirror of the other galvano scanner.
The laser light emerged from the optical scanner 103 is incident on the dichroic mirror 106 through the pupil projection lens 104 and the image forming lens 105. The laser light is reflected at the dichroic mirror 106, and is incident on the microscope objective 107 (hereinafter, appropriately called as ‘objective 107’). The laser light is converged by the objective 107, and a laser spot is formed on a specimen 108.
The laser spot moves two-dimensionally on the specimen 108 according to the movement of mirrors of the galvano scanners. Accordingly, scanning of the specimen 108 is carried out. Positions of the mirrors of the galvano scanners and a position of a pupil 107a of the objective 107 are substantially conjugate through the pupil projection lens 104 and the image forming lens 105.
In a case in which, the specimen 108 is a fluorescent specimen, fluorescent light is emitted from the specimen 108. The fluorescent light passes through the objective 107 and the dichroic mirror 107, and is incident on the photodetector 109. The fluorescent light incident on the photodetector 109 is converted to an electric signal by the photodetector 109. By performing a sampling of the electric signal output from the photodetector 109 in synchronization with scanning, a fluorescence image of the specimen 108 is acquired.
An illustration of as to how the light stimulus is applied is shown in FIG. 9A, FIG. 9B, and FIG. 9C. FIG. 9A is a diagram showing an appearance of an actual field of view, FIG. 9B is a diagram showing a relationship of a position of a cell, a timing of changing the light intensity, and the light intensity, and FIG. 9C shows an image of an observed object when the light intensity has been changed.
In FIG. 9A, a rectangular area 221 in an actual field of view 220 of a microscope is a scanning area. Cells in the rectangular area 221 are scanned and subjected to imaging. A plurality of cells exists in the rectangular area 221, and here, light stimulus is to be applied to two cells namely a cell A and a cell B.
In FIG. 9B, a cross-sectional view of the cells along a line XX′ in FIG. 9A is shown. Moreover, beneath the cross-sectional view, a graph indicating a change in the light intensity in scanning of one line is shown. A vertical axis of the graph is a light intensity I and a horizontal axis of the graph is a scanning time t. As shown in FIG. 9B, the light intensity of laser light irradiated to the cell A and cell B is, 50% of the maximum light intensity for the cell A, and 10% of the maximum light intensity for the cell B. Moreover, scanning of one line is carried out in 1 msec. Therefore, the light intensity is to be changed to 10% or 50% in 1 msec.
In the acoustic optical element 102, the amplitude of the high-frequency voltage applied is changed according to the time elapsed from a start point of scanning of one line (scanning position). Accordingly, an arrangement is made such that laser light of various light intensities are irradiated to the cell A and the cell B.